Individual phosphorylation sites at the C-terminus of the apelin receptor play different roles in signal transduction

The apelin and Elabela proteins constitute a spatiotemporal double-ligand system that controls apelin receptor (APJ) signal transduction. Phosphorylation of multiple sites within the C-terminus of APJ is essential for the recruitment of beta-arrestins. We sought to determine the precise mechanisms by which apelin and Elabela promote APJ phosphorylation, and to elucidate the influence of beta-arrestin phosphorylation on G-protein-coupled receptor (GPCR)/beta-arrestin-dependent signaling. We used techniques including mass spectrometry (MS), mutation analysis, and bioluminescence resonance energy transfer (BRET) to evaluate the role of phosphorylation sites in APJmediated G-protein-dependent and beta-dependent signaling. Phosphorylation of APJ occurred at five serine residues in the C-terminal region (Ser335, Ser339, Ser345, Ser348 and Ser369). We also identified two phosphorylation sites in beta-arrestin1 and three in beta-arrestin2, including three previously identified residues (Ser412, Ser361, and Thr383) and two new sites, Tyr47 in beta-arrestin1 and Tyr48 in beta-arrestin2. APJ mutations did not affect the phosphorylation of beta-arrestins, but it affects the beta-arrestin signaling pathway, specifically Ser335 and Ser339. Mutation of Ser335 decreased the ability of the receptor to interact with beta-arrestin1/2 and AP2, indicating that APJ affects the beta-arrestin signaling pathway by stimulating Elabela. Mutation of Ser339 abolished the capability of the receptor to interact with GRK2 and beta-arrestin1/2 upon stimulation with apelin-36, and disrupted receptor internalization and beta-arrestin-dependent ERK1/2 activation. Five peptides act on distinct phosphorylation sites at the APJ C-terminus, differentially regulating APJ signal transduction and causing different biological effects. These findings may facilitate screening for drugs to treat cardiovascular and metabolic diseases.